Treatment with dialkoxy pyridopyrimidines

ABSTRACT

Potent antiproliferative activity in combination with low inhibition of histamine N-methyltransferase has been found in a class of 2,4-diamino-6-(2,5-dialkoxybenzyl)-5-methylpyrido[2,3-d]pyrimidines.

This application is a continuation of application Ser. No. 384,147,filed June 1, 1982, now abandoned, which is a division of Ser. No.228,164, filed Jan. 23, 1981, now U.S. Pat. No. 4,372,957, which is acontinuation of Ser. No. 159,243, filed June 13, 1980, now abandoned.

The present invention relates to 2,4-diaminopyrido(2,3-d)pyrimidines, topharmaceutical formulations comprising such compounds and to their usein medicine.

U.K. Pat. No. 1 084 103 discloses 2,4-diaminopyrido(2,3-d) pyrimidinesof the general formula (I): ##STR1## in which R¹ is an alkyl group andR² is an unsubstituted benzyl group or a benzyl group substituted by oneor more halogen atoms, alkyl or alkoxy groups.

The compounds of formula (I) were described as having high in vitro andin vivo activity against bacteria or bacterial infections inexperimental animals.

Subsequently it has been found that the compounds of formula (I)specifically disclosed in U.K. No. 1 084 103 show some inhibitoryactivity against mammalian dihydrofolate reductase (DHFR), and theactivity was sufficient to render them potentially useful in thetreatment of conditions where inhibition of mammalian DHFR is desirable.It has further been found that many of these compounds are potentinhibitors of histamine N-methyl-transferase (HMT), an enzyme involvedin the metabolism of bistamine. In this manner they often cause anundesirable accumulation of bistamine in organs and tissues. The effectsof bistamine are well known and any possibility of a further utility forthese compounds was substantially diminished by their strong inhibitionof HMT.

Further investigation showed that a number of other compounds of formula(I) also possess DHFR inhibitory activity but that these, too, were alsopotent inhibitors of HMT. Others, which has acceptably low levels ofinhibition of HMT were found to have insufficient activity as inhibitorsof DHFR.

It has now been surprisingly found that2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine,which is within the scope of formula (I) but not specifically disclosedin U.K. No. 1 084 103, is not only a very potent inhibitor of mammalianDHFR, but also has acceptably low inhibitory activity against HMT. Thiscompound is represented by formula (II) below and is useful in thetreatment of proliferative diseases, such as psoriasis basal andsquamous cell carcinomas of the skin, and various forms of cancerincluding leukemias, lymphomas, sarcomas and solid tumors.

The invention herein accordingly sets forth the antiproliferativeproperties of the compound in formula (II): ##STR2## wherein R and R'are lower (C₁ -C₆) alkyl and pharmaceutically acceptable acid additionsalts thereof. Preferably mono-basic salts are provided. Preferably Rand R' are methyl.

The compounds of formula (II) and their use in the treatment ofinfections caused by Streptoccous pyogenes and Streptacoccus faecaliswere discovered earlier by another whose rights are assignable toBurroughs Wellcome Co.

The antiproliferative activity of the compound of formula (II) residesin the free base and thus the nature of the acid participating in theacid addition salts is of minor importance. Such acid addition saltsinclude, for example, those derived from hydrochloric acid, hydroiodiccid, sulphuric acid, phosphoric acid, acetic acid, p-toluenesulphonicacid, methanesulfonic acid, maleic acid, lactic acid, citric acid,tartartic acid, succinic acid, oxalic acid, p-chlorobenzenesulphonicacid, isethionic acid, glucuronic acid. pantothenic acid and lactobionicacid.

The compound of formula (II) may be prepared by any method known in theart for the preparation of compounds of analogous structure.

In particular the compound of formula (II) may be prepared by thereductive cleavage of the corresponding 7-substituted compounds of theformula (III): ##STR3## wherein R and R' are as defined above and R³ isa leaving group capable of being removed by hydrogenolysis. Such groupsinclude for example a mercapto or a halogeno (e.g. chloro) group.

Where in compound (III) R³ is SH the dethiation may for instance beconveniently effected by reaction with a reducing agent, for exampleRaney nickel or Raney cobalt or by catalytic hydrogenation utilizedhydrogen in the presence of a catalyst such as palladium on charcoal.

The compound of formula (III) wherein R³ is a mercapto group may beprepared from the corresponding 7-chloro compound (III) [R³ ═Cl], byreaction with a hydrosulfide as described in U.K. Pat. No. 913 710 or bytreatment of the corresponding 7-hydroxy compound with phosphoruspentasulfide.

In the case where R³ is a halogen atom in the compound of formula (III)the compound of formula (II) may for instance be conveniently obtainedby e.g. catalytic hydrogenation.

The compound of formula (II) may also be prepared by reacting2,4,6-triaminopyrimidine (IV) with a compound of formula (V): ##STR4##wherein A is selected from ##STR5## R and R' are as defined above; andR⁴ is a leaving group such, for example, as a tertiary amino, alkoxy,alkylthio, halogeno, sulphonate or tosylate group.

The compound of formula (II) may additionally be prepared by theconversion to amino groups, by methods known in themselves in pyrimidinechemistry, of the hydroxy and/or mercapto group(s) in the compound offormula (VI): ##STR6## in which Y and Z are the same or different andare OH, SH or NH₂ provided that at least one of Y and Z is OH or SH.

Compounds of formula (VI) may be prepared by methods known in the artfor the preparation of such compounds. In addition, those in which Y isOH or SH may be obtained for example by reaction of urea, guanidine orthiourea with a suitable compound of formula (VII): ##STR7## in which Rand R' are as defined above and R⁵ is --CO₂ H, CO₂ Alkyl, CONH₂ or CNand R⁶ is NH₂, Cl or Br.

While it is possible for a compound of formula (II) or an acid additionsalt thereof (hereinafter referred to as the "active compounds") to beadministered as the raw chemical it is preferably presented in the formof a pharmaceutical formulation.

The invention therefore further provides a pharmaceutical formulationcomprising the active compound together with a pharmaceuticallyacceptable carrier therefore. The carrier must be "acceptable" in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

The invention additionally provides a method for the preparation of apharmaceutical formulation comprising bringing into association anactive compound and pharmaceutically acceptable carrier therefor.

Topical application is particularly suitable when the active compoundsare for use in the treatment of proliferative skin diseases.

The term "topical" as applied herein relates to the use of the activeingredient incorporated in a suitable pharmaceutical carrier, andapplied at the site of the disease for the exertion of local action.

Pharmaceutical formulations suitable for topical administration may bepresented in anhydrous forms such as ointments, lotions, pastes,jellies, sprays, aerosols, and bath oils. The term ointment includesformulations (including creams) having oleaginous, absorption,water-soluble and emulsion type bases, for example petrolatum, lanolin,polyethylene glycols and mixtures thereof.

Topical formulations may contain a concentration of the activeingredient of from about 0.05 to about 2% w/w, preferably about 0.1 toabout 1% w/w, most preferably about 0.2 to about 0.5% w/w.

Other pharmaceutical formulations include those suitable for oral,rectal, and parenteral administration although of those oral ispreferred. The formulations may, where appropriate, be convenientlypresented in discrete dosage units and may be prepared by any of themethods well known in the art of pharmacy. A convenient unit doseformulation contains the active compound in amount of from about 50 mgto about 1 g, preferably about 100 mg to about 500 mg, most preferablyabout 200 mg, to be taken once or several times daily.

All methods for the preparation of such formulations include the step ofbringing into association the active compound with liquid carriers orfinely divided solid carriers or both and then, if necessary, shapingthe product into the desired formulation.

Pharmacutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules, cachets or tablets eachcontaining a predetermined amount of the active compound. A tablet maybe made by compression or moulding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active compound in a free-flowing form such asa powder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating, surface active or dispersing agent. Mouldedtablets may be made by moulding an inert liquid diluent. Tablets may beoptionally coated and, if uncoated, may be optionally scored. Capsulesmay be prepared by filling the active compound ingredients, into thecapsule cases and then sealing them in the usual manner. Cachets areanalogous to capsules wherein the active ingredient together with anyaccessory ingredient(s) are sealed in a rice paper envelope.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a liquid may be presented as a solution or a suspension in anaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and other materialcommonly used in the art, and the suppositories may be convenientlyformed by admixture of the active compound with the softened or meltedcarrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of the active compound inaqueous or oleaginous vehicles. Such preparations are convenientlypresented in unit dose or multi-dose containers which are sealed afterintroduction of the formulation until required for use.

It should be understood that in addition to the aforementioned carrieringredients the pharmaceutical formulations described above may include,as appropriate, one or more additional carrier ingredients such asdiluents, buffers, flavouring agents, binders, surface active agents,thickeners, lubricants, preservatives (including anti-oxidants) and thelike, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

As has been described above the compounds of the present invention areuseful for the treatment of proliferative diseases. The invention thusfurther provides a method for the treatment of a proliferative diseasein mammals which comprises the administration of an effective, non-toxicamount of the compound of formula (II) or an acid addition salt thereof,once or several times a day orally, or applied topically.

The amount of compound of formula (II) required for therapeutic effect san antiproliferative agent will of course vary not only with theparticular salt used but also with the route of administration. Ingeneral, a suitable dose for the treatment of mammals will lie in therange of from about 0.1 to about 100 mg per kilogram bodyweight (mg/kg)per day, preferably in the range from of about 2.0 to about 50 mg/kg,more preferably in the range of about 0.5 to about 20 mg/kg, mostpreferably in the range of about 1 to about 10 mg/kg.

Toxic manifestations attributable to the active compound are typicallythose associated with folate depletion, such as bone marrow depression,megaloblastic changes, and gastrointestinal ulceration. Calciumleucovorin (calcium salt of 5-formyl-5,6,7,8-tetrahydrofolic acid) maybe administered to effect reversal of these toxic manifestations or toprevent their occurrence. The administration of calcium leucovorin maybe effected concurrently with treatment or at any stage thereof whenevertoxic symptoms appear.

Thus, the haemtological activity of the active compound can be preventedor reduced by the simultaneous administration of leucovorin.Consequently, tissue levels of the active compound may be safely raisedby increasing the dose of the compound together with a simultaneousadministration of leucovorin.

The following Examples, which illustrate the invention, should in no waybe construed as constituting a limitation thereof.

EXAMPLE 12,4-Diamino-5-methyl-6-(2,5-dimethoxybenzyl)pyrido[2,3-]pyrimidine

A mixture of 2,5-dimethoxybenzaldehyde (100 g), ethyl acetoacetate (84.5g) and anhydrous benzene (200 ml), piperidine (6 ml) and acetic acid (12ml) was heated at reflux for 3 hours in an apparatus fitted with aDean-Stark trap to collect the azeotropically distilled water. Thereaction mixture was cooled, benzene (300 ml) added, and the solutionwas washed successively with water (100 ml), cold 0.1N hydrochloric acid(200 ml), 5% aqueous sodium bicarbonate (200 ml) and dilute acetic acid(100 ml) and dried over anhydrous magnesium sulfate. The solvent wasthen removed under reduced pressure and the residual oil distilled, b.p.169°-170° C./0.3 mm Hg. The product, ethylα-(2,5-dimethoxybenzylidene)acetoacetate, solidified on standing (104 g,m.p. 60-69° C.) and was recrystallized from ethanol-pentane (m.p.72°-73° C.). A portion (38 g) of the product was reduced catalyticallyin the presence of palladium on charcoal catalyst (Pd/C) in ethylacetate (150 ml). The product, afer removal of solvent, was purified bydistillation under reduced pressure to give ethylα-(2,5-dimethoxybenzyl)acetoacetate,b.p. 146°-148° C./0.3 mm Hg.

A mixture of ethyl α-(2,5-dimethoxybenzyl)acetoacetate (21.2 g),2,4,6-triaminopyrimidine (10 g) and diphenyl ether (100 ml) was heatedat 190°-230° C. for 1.5 hours in an apparatus fitted with a Dean-Starktrap and water-ethanol (4 ml) was collected. Methanol (200 ml) andethanel (50 ml) were added to the cooled reaction mixture. The resultingsolid was collected by filtration and treated with boiling water (1 l)to give2,4-diamino-5-methyl-6-(2,5-dimethoxybenzyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidine (17 g), m.p. 325°-326° C.

2,4-Diamino-5-methyl-6-(2,5-dimethoxybenzyl)-7-oxo-7,8-dihydropyrido[2,3-d]dipyrimidine (8g) was chlorinated by treatment with Vilsmcier reagent prepared byslowly adding thionyl chloride (28.6 ml) in dry chloroform (25 ml) to asolution of dimethylformamide (17.5ml) in chloroform (100 ml) at 0°-5°C. The cold mixture of the pyridopyrimidine and Vilsmcier reagent wasstirred, gradually allowed to reach ambient temperature and then heatedat reflux for 3 hours. It was then treated with ethanolic base (80 ml)maintaining the temperature at 25°-30° C. with cooling. The brownproduct formed was isolated, treated further with aqueous ammonia andthen recrystallized from ethanol to give2,4-diamino-5-methyl-6-(2,5-dimethoxybenzyl)-7-chloro-pyrido[2,3-d]pyrimidine,m.p. 193°-196° (dec.).

The chloro compound (0.3 g) was dissolved in ethanol (200 ml) containingpotassium hydroxide (0.2 g). Palladium on charcoal catalyst (0.2 g) wasadded and hydrogenation commenced. Reduction was complete after 48 hoursand yielded2,4-diamino-5-methyl-6-(2,5-dimethoxybenzyl)pyrido[2,3-d]pyrimidine,m.p. 252°-254° C.

EXAMPLE 2 Inhibition of Mammalian Dihydrofolate Reductase (DHFR) by2,4-Diamino-5-methyl-6-benzylpyrido(2,3-d)pyrimidnes

The inhibitory effect of the test compounds against DHFR partiallypurified from rat liver was determined. The results are given in Table 1below. An IC₅₀ of 5×10 ⁻⁸ M or less is considered significant potency,an IC₅₀ of 1×10⁻⁸ M or less being particularly significant potency.

                  TABLE 1                                                         ______________________________________                                        TEST COMPOUND                                                                  ##STR8##                                                                     R.sup.1 R.sup.2           IC.sub.50 × 10.sup.-8 M                       ______________________________________                                        CH.sub.3                                                                               ##STR9##         4                                                   CH.sub.3                                                                               ##STR10##        1.6                                                 CH.sub.3                                                                               ##STR11##        2.7                                                 CH.sub.3                                                                               ##STR12##        5.0                                                 CH.sub.3                                                                               ##STR13##        5.3                                                 CH.sub.3                                                                               ##STR14##        9                                                            ##STR15##        25                                                  CH.sub.3                                                                               ##STR16##        0.15                                                ______________________________________                                    

EXAMPLE 3 Inhibition of Histamine N-Methyltransferase (HMT) by2,4-Diamino-5-methyl-6-benzylpyrido-(2,3-d)pyrimidines

The effect of the test compounds used in Example 3 against HMT wasdetermined. The results are given in Table 2. An inhibition of less than20% was considered acceptable and the lack of any effect on this enzymeis most desirable since any interference with histamine metabolismshould be minimized.

                  TABLE 2                                                         ______________________________________                                        TEST COMPOUND                                                                  ##STR17##           % Inhibition of HMT                                      R.sup.1                                                                             R.sup.2           at 10.sup.-5 M                                        ______________________________________                                        CH.sub.3                                                                             ##STR18##        51                                                    CH.sub.3                                                                             ##STR19##        59                                                    CH.sub.3                                                                             ##STR20##        18                                                    CH.sub.3                                                                             ##STR21##        20                                                    CH.sub.3                                                                             ##STR22##        55                                                           ##STR23##        48                                                    CH.sub.3                                                                             ##STR24##        11                                                    ______________________________________                                    

EXAMPLE 4 Antitumor Effect of2,4-Diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido(2,3-d)-pyrimidineAgainst a Solid Tumor (Carcinoma)

Solid Walker 256 tumors were implanted subcutaneously into three groupsof rats. On the third day after implantation with the tumor, drugtreatment was commenced and continued for 5 successive days. One groupof rats was treated with2,4-diamino-6-(2,5-dimethoxyphenyl)-5-methylpyrido[2,3-d]pyrimidine at adose of 25 mg/kg q.i.d., a second group was treated with the samecompound at 15 mg/kg b.i.d.; the third group was left untreated ascontrols. After 17 days the treated groups of rats showed mean tumorvolumes of 29% and 10% respectively of the untreated group.

In a second study tumors were implanted subcutaneously and drugtreatment delayed until 10 days after implantation. Drug treatmentconsisted of 30 mg/kg q.i.d. on days 10-13 and 50 mg/kg on days 20, 24and 28 after tumor implantation. Mean tumor volumes were measured duringthe course of their study. Tumor volumes in animals which received notreatment increased rapidly whereas those in animals which received drugtreatment showed a decrease in mean tumor volume during the period ofobservation. Twenty-three days after tumor implantation the T/C was0.03.

EXAMPLE 5 Antileukemic Effect of2,4-Diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido(2,3-d)pyrimidine

Leukemia P388 cells were inoculated intraperitoneally of CDF, CharlesRiver female mice, each animal receiving 10⁶ cells. On the second dayafter implantation with the leukemia cells, drug treatment was commencedand continued twice daily for three days. Four groups of mice weretreated with2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido(2,3-d)pyrimidine atdoses of 22, 33.5, 50 and 75 mg/,g of body weight and a fifth group wasleft untreated as a control. All of the untreated mice died from theleukemia between the 10th and 12th day with a median survival of 11days. All of the treated mice survived for periods longer than theuntreated animals with a % increase in life span of 145%, 159% 140% and154% for the drug treated groups receiving 22, 33.5, 50 and 75 mg/kgdoses, respectively. This is a highly significant effect and there wasno evidence of drug-related toxicity.

Within the same experiment, two closely related compounds were testedfor antileukemic activity at the same dosage levels of 22, 33.5, 50 and75 mg/kg, respectively, and the same schedule of treatment. Thecorresponding pyridopyrimidine with a 6-(4-methoxybenzyl)substituent wasinactive at all doses and the 6-(2-chlorobenzyl) compound had onlymarginal or slight activity. Thus, the 6-((2,5-dimethoxybenzyl)compoundhas substantially greater antileukemic activity than these comounds ofsimilar structure.

EXAMPLE 6 Water Soluble Ointment

    ______________________________________                                                              Amount (g)                                              ______________________________________                                        2,4-Diamino-5-methyl-6-(2,5-                                                                          0.5                                                   dimethoxybenzyl)pyrido[2,3-d]pyrimidine                                       Polyethylene glycol 300 20.0                                                  Polyethylene glycol 1500                                                                              79.5                                                  Total                   100.0                                                 ______________________________________                                    

EXAMPLE 7 Skin Cream

    ______________________________________                                                              Amount (g)                                              ______________________________________                                        2,4-Diamino-5-methyl-6-(2,5-                                                                          0.5                                                   dimethoxybenzyl)pyrido[2,3-d]pyrimidine                                       Glyceryl monostearate   20.0                                                  Methylparaben           0.3                                                   Petrolatum, light liquid                                                                              4.0                                                   Propylene glycol        5.0                                                   Span 60                 2.0                                                   Tween 61                4.0                                                   Water                   64.2                                                  Total                   100.0                                                 ______________________________________                                    

EXAMPLE 8 Injectable

    ______________________________________                                                               Amount                                                 ______________________________________                                        2,4-Diamino-5-methyl-6-(2,5-                                                                           qs to 5 mg/ml                                        dimethoxybenzyl)pyrido[2,3-d]pyrimidine                                       Propylene glycol         40 ml                                                Ethanol                  11 ml                                                Water                    40 ml                                                ______________________________________                                    

EXAMPLE 9 Injectable

    ______________________________________                                                               Amount                                                 ______________________________________                                        2,4-Diamino-5-methyl-6-(2,5-                                                                           qs to 5 mg/ml                                        dimethoxybenzyl)pyrido[2,3-d]pyrimidine                                       Propylene glycol         40 ml                                                5% Dextrose solution     60 ml                                                ______________________________________                                    

We claim:
 1. The method of prolonging the life of a mammal sufferingfrom leukemia which comprises administering to said mammal an effectiveantileukemia amount of the compound2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyridopyrimidine or apharmaceutically acceptable acid addition salt thereof together with apharmaceutically acceptable carrier.
 2. The method of treating carcinomain a mammal suffering from a carcinoma comprising the administration tosaid mammal of a nontoxic, effective anticarcinoma treatment amount of2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyridopyrimidine or apharmaceutically acceptable acid addition salt thereof together with apharmaceutically acceptable carrier to said mammal.
 3. The method oftreating sarcoma in a mammal suffering from a sarcoma which comprisesadministering to said mammal a nontoxic, effective antisarcoma amount of2,4-diamino-6-(2,5-dimethoxybenzyl-5-methylpyridopyrimidine or apharmaceutically acceptable acid addition salt thereof to said mammal.4. The method of treating leukemia in a mammal suffering from leukemiawhich comprises administering to said mammal a nontoxic, effectiveantileukemia amount of2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyridopyrimidine or apharmaceutically acceptable acid addition salt thereof to said mammal.5. A method of decreasing the volume of a carcinoma growing in a mammalcomprising the administration to said mammal of an effective carcinomacolume decreasing amount of the compound2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyridopyrimidine or apharmaceutically acceptable acid addition salt thereof.